B. Protein Blotting

A general protocol for sample preparation.

Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

(Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.

Wash membrane again four times for 5 min each in TBST.

The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

Source / Purification

MultiMab™ rabbit monoclonal mix antibodies are prepared by combining individual rabbit monoclonal clones in optimized ratios for the approved applications. Each antibody in the mix is carefully selected based on motif recognition and performance in multiple assays. Each mix is engineered to yield the broadest possible coverage of the modification being studied while ensuring a high degree of specificity for the modification or motif.

Background

Apoptosis is a physiological process resulting in a highly regulated, programmed form of cell death that is a normal part of growth and development in multicellular organisms. Caspases are aspartic acid-directed cysteine proteases that are central to the apoptotic mechanism (1). The intrinsic pathway initiates an apoptotic cascade from signals originating within the cell, such as DNA damage, while an extrinsic pathway initiates apoptosis in response to extracellular signals, like FasL. In both intrinsic and extrinsic pathways, initiator caspases cleave downstream substrates, including multiple effector caspases and the primary executioner of cell death, caspase-3 (2,3). Effector caspases amplify the apoptotic cascade to target many critical proteins needed for normal cell function. Apart from its role in developmental biology, the regulation of apoptosis has broad implications for the study of cancer, autoimmune disorders and infectious diseases (4). Thousands of known and putative caspase cleavage sites are present within the human proteome; almost all sites involve cleavage at an aspartic acid residue, though cleavage at glutamic acid residues is seen, rarely, as well (5).